Kidney cell function requires the maintenance of polarized plasma membrane domains with distinct protein and lipid compositions. This is accomplished in part by the vectorial delivery of newly synthesized membrane proteins to the apical and basolateral surface. These proteins traffic together through the Golgi, and but emerge from the trans-Golgi network (TGN) in distinct populations of carriers. Surprisingly little is known about how apically destined proteins are selected and packaged in the TGN, although association with glycolipid-enriched lipid microdomains is thought to be important for the delivery of a subset of apical proteins. In addition, there is accumulating evidence that both N- and O-glycans can function as apical targeting motifs on some proteins. This proposal is designed to address the following questions: 1. Do apical proteins that are sorted via distinct mechanisms exit the TGN in common carriers or in separate vesicles? 2. What cytoplasmic components are required for the export of apical proteins from the TGN? and 3. How is the sorting of proteins that require N-glycans for apical delivery accomplished? Our working hypothesis is that some aspects of apical cargo loading and vesicle formation will resemble known mechanisms that operate along the biosynthetic and endocytic pathways. We will use in vitro assays and live-cell imaging to examine the transport requirements for distinct classes of apical proteins: these approaches in combination will provide a more comprehensive view of how apical protein export from the TGN is regulated. [unreadable] [unreadable]